A poorly understood collagen post-translational modification has been 3-prolyl-hydroxylation (P3H) converting proline to 3-hydroxy proline (3-Hyp). It occurs uniquely in the fibrillar collagens, i.e., types I, II and III collagen, at only one proline position, but abundantly in other collagens such as network collagens, i.e., type IV collagen, where it occurs in up to 10% of residues. This begs the question of whether 3-Hyp residues may serve divergent biological functions in different settings and tissues. In recent data, we have identified a novel protein, CRTAP or Cartilage Associated Protein, that is representative of the Leprecanfamily of proteins recently reported to contain a conserved 2-oxoglutarate dioxygenase domain that is found in collagen 4-prolyl-hydroxylases, Hypoxic Inducible Factor (HIF) 4-prolyl-hydoxylases (PDHs), and lysyl hydroxylases (PLODs). Moreover, Leprecan or P3H1 has collagen 3-prolyl-hydroxylase activity in vitro implicating this family of genes as the long sought after PSH's. By combining human and mouse genetic, and proteomic approaches, we show (see Preliminary Studies C.2.) that loss of Crtap in mice causes an osteochondrodysplasia characterized by short stature, kyphosis, and severe osteoporosis. Moreover, this phenotype is biochemically associated with conversion of the single 3-hydroxy- proline to proline in the triple helical domain of types I and II collagen. CRTAP can bind P3H1 and is required for P3H activity in vivo. Finally, we found that loss of CRTAP is associatedwith recessive osteogenesis imperfecta types VII and ll/lll in humans. These data raise important questions that we will address in our Specific Aims. 1) Are the phenotypic features of Crtap loss of function due solely to loss of 3-prolyl- hydroxylation of fibrillar collagens and are there redundant functions with P3H1? 2) What are the consequences of 3-Hyp loss in cartilage and bone on cellular differentiation and function, and collagen biosynthesis? 3)What is the full human clinical spectrum associated with loss of 3-prolyl-hydroxylation of fibrillar collagens? By combining mouse and human studies, this proposal will interact directly with Projects I, II, and the Protein Biochemistry Core to answer these questions.